Spectroscopic tools based on emission and detection of an optical signal are widely used to characterise, measure and/or detect components of a sample on the spot. Non-limiting examples of these optical signals are fluorescence, phosphorescence, Rayleigh scattering, Raman scattering and atomic emission signals. These tools offer the ability to measure directly on a defined spot in a non-contact fashion.
Raman analysis is based on inelastic scattering of excitation light by a sample to produce a spectrum of scattered light that is characteristic of the sample. The spectral lines are dependent on vibrational motion in the sample components and the probabilities of scattering. A sample consisting of a mixture of components results in a spectrum that is a linear combination of the component spectra. Hence, relative chemical content can be determined in a spectroscopic measurement using appropriate analysis of the spectrum. For more information on the nature of Raman spectroscopy the reader is referred to standard textbooks on Raman spectroscopy.
Conventional Raman spectroscopy is performed in backscatter mode wherein a sample spot is irradiated with excitation light and the backward scattered radiation is detected in the direct vicinity of the excitation spot. In a backscatter mode, the excitation beam and the resulting scattered optical signal travel through the same optical system, with optical splitting optics used to separate the emitted optical signal beam from the excitation signal beam, for example based on their different frequencies. Transparent or semi-transparent materials allow samples to be characterized beneath the sample surface (i.e. into the depth of the material of the sample) by performing measurements in the z-direction. However, although this type of depth analysis of samples is also desired when the material of interest is buried under a semi-, or non-transparent material such as a sheet of paper or a plastic cup for example, the conventional backscatter measurement is limited to the near-surface of such diffusely scattering objects. For example, with tissue it is limited to the first few hundred micrometers depth of surface material. Hence depth measurements are shielded and virtually impossible for non-transparent samples. The origin of this limitation is that the excitation signal intensity is high in the region of excitation so that it dominates the collected scattered radiation signal.
Spatially Offset Raman Spectroscopy (SORS) is a measurement variant that solves the above limitation in that it allows highly accurate chemical analysis of objects beneath obscuring surfaces, including for example tissue, coatings and packaging materials such as bottles. Examples of the fields of use of this SORS method include analysis of: bone beneath skin, contents inside plastic bottles for quality or composition control, security measurements as with detection of explosives inside containers and counterfeit practices such as with tablets inside blister packs.
The basic SORS method makes use of the fact that most sample materials are neither completely transparent to light, nor completely block it. Instead, they tend to scatter the excitation light much the same as when a red laser pointer illuminates the end of a finger, the light scatters throughout a large part of the tissue in the finger. Wherever the excitation light arrives in the sample, there will occur some inelastic scattering due to the Raman effect if Raman active materials are present in the sample. Thus, most parts of a sample, although not directly excited by the excitation spot, will generate a Raman signal, even if it is not at the surface of the sample. The SORS measurement is set up such that it avoids detection of scattered radiation at the dominating excitation region. Thus, more particularly, the SORS method is based on the collection of Raman spectra from regions away from the point of excitation on the sample surface, i.e. from regions that are spatially offset with regard to this excitation region. The spectra for laterally offset regions contain different relative contributions from sample layers located at different depths (z-direction) in the sample material. This difference is brought about by a wider lateral diffusion of photons emerging from greater depths of the sample.
Thus, by making at least two Raman measurements, one at the surface and one at an offset position of typically a few millimeters away and by subtracting these spectra using a scaled subtraction, two spectra can be produced of which one represents the subsurface (interior of the sample) and the other represents the surface. For a simple two-layer system, such as powder in a plastic bottle, the powder spectrum can be measured without knowing the bottle material or its relative signal contribution. To do this without using an offset measurement would be severely restricted by photon shot noise generated by Raman and fluorescence signals originating from the surface layer.
A further useful sub-variant of SORS that improves certain measurements such as analysis of tissue in vivo is Inverse SORS. Rather than use a spot collection geometry and a circular spot for illumination, the constant offset is maintained by exciting the sample with a ring of light centred on the collection region.
Although scaled subtraction of spectra works well for two-layer samples, samples with more complicated compositions, such as where the overlying material contains components included in the sub-layer as in living tissue, for example, may require multi-variate analysis (e.g. Principal Component Analysis). This in turn means however that it is necessary to take several spectra at different offset distances. In the different spectra, as the spatial offset increases, also the ratio of the spectral contribution sub-surface/surface increases, providing again a separation of components using the multi-variate analysis. The limit for this is given by the fact that the total detected signal also decreases with increasing offset, so that the maximum has to be offset against signal to noise ratio in a practical measurement.
The devices capable of measuring SORS spectra rely on complicated excitation detection devices often in combination with movable stages for measuring the spectra at variable offsets. Such devices are difficult to use in application fields where measurements are performed on the spot.
In conventional Raman spectrometers the locations of excitation and detection are fixed by the optical system and in most systems these locations exactly overlap (backscattering configuration). Therefore it is difficult to implement SORS in conventional Raman systems without costly and extensive redesigns. Hence there is a need for simplified equipment that is robust yet does provide the flexibility to perform the offset measurements at variable offsets.